Chagas disease afflicts over 12 million people in Central and South America. Eradication efforts directed at the triatomine vectors of this disease have been effective, but are fraught with issues of cost, toxicity of pesticides and vector resistance. A paratransgenic strategy is being developed as an adjunctive approach. Rhodococcus rhodnii, the symbiont of Rhodnius prolixus, has been transformed to express cecropin A, an anti-trypanosomal peptide. Parasite-refractory R. prolixus have been developed in the lab and a single chain antibody has been expressed in these insects. Strategies for spreading transgenic symbionts into field vectors and detailed risk assessment are under development. Field use will require an armamentarium of molecules that target multiple sites on T. cruzi. This 4 year application aims to develop 3 broad classes of molecules with anti- trypanosomal activity. The first of these, addressed in Aim 1, are single chain antibodies (scFv). The surface glycoprotein, gp72, an integral part of T. cruzi maturation, is the first target for scFv development. In Aim 2, immune peptides related to cecropin A- magainin, moricin, mellitin and Apidaecin will be tested for activity against T. cruzi. In Aim 3, mannosidase and lyticase that target the surface sugar moieties of T. cruzi will be developed as effector molecules. cDNA of all classes of molecules will be expressed in the bacterium R. rhodnii to assess selective toxicity to T. cruzi and effects on bacterial physiology. In Aim 4, potential toxicity of transgenic R. rhodnii to R. prolixus will be determined. Furthermore, strategies to deliver combinations of transgenic bacteria concurrently will be explored. This project aims to develop multiple lines of transgenic bacteria expressing anti- trypanosomal molecules, to be used for future field deployment in the ongoing battle against Chagas disease. The Rhodnius prolixus system will potentially have application in a defined geographical area but will also serve as a model for paratransgenic control of Chagas disease, in other endemic regions, with other triatomine vectors of Chagas disease.